Mechanisms of Leukocyte Distribution During Sepsis
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Ploppa et al. Critical Care 2010, 14:R201 http://ccforum.com/content/14/6/R201 RESEARCH Open Access Mechanisms of leukocyte distribution during sepsis: an experimental study on the interdependence of cell activation, shear stress and endothelial injury Annette Ploppa1, Volker Schmidt1, Andreas Hientz2, Joerg Reutershan1, Helene A Haeberle1, Boris Nohé1* Abstract Introduction: This study was carried out to determine whether interactions of cell activation, shear stress and platelets at sites of endothelial injury explain the paradoxical maldistribution of activated leukocytes during sepsis away from local sites of infection towards disseminated leukocyte accumulation at remote sites. Methods: Human umbilical venous endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) were activated with lipopolysaccharide at 100 and 10 ng/ml to achieve adhesion molecule patterns as have been reported from the hyper- and hypo-inflammatory stage of sepsis. To examine effects of leukocyte activation on leukocyte-endothelial interactions, activated HUVEC were perfused with activated and non-activated neutrophils in a parallel plate flow chamber. Adhesion molecule expression and function were assessed by flow cytometry and blocking antibodies. In a subset of experiments the sub-endothelial matrix was exposed and covered with platelets to account for the effects of endothelial injury. To investigate interactions of these effects with flow, all experiments were done at various shear stress levels (3 to 0.25 dyne/cm2). Leukocyte-endothelial interactions were analyzed by videomicroscopy and analysis of covariance. Results: Activation of neutrophils rendered adhesion increasingly dependent on shear stress reduction. At normal shear stress, shedding of L-selectin decreased adhesion by 56%. Increased rolling fractions of activated PMN at low shear stress revealed impaired integrin affinity despite numerical up-regulation of CD11b. On sub-maximally activated, intact HUVEC shear stress became the prevailing determinant of adhesion. Presence of a platelet-covered injury with high surface density of P-selectin was the strongest variable for adhesion. When compared to maximally activated HUVEC, platelets increased neutrophil adhesion by 2.7-fold. At sub-maximal activation a 10-fold increase was observed (P < 0.05 for all). Conclusions: L-selectin shedding and integrin dysfunction render leukocyte adhesion increasingly susceptible to shear stress and alternative adhesion receptors. In combination, these effects inhibit recruitment to normally perfused sites with intact endothelium and favor maldistribution towards sites with compromised perfusion or endothelial injury. Introduction upregulation of endothelial adhesion molecules in Directing leukocytes to local sites of infection is a cru- inflamed tissue, resulting in a targeted accumulation of cial part of the innate immune response. While intravas- leukocytes at the site of infection [1]. Initially, selectin- cular shear forces prevent relevant leukocyte adhesion in dependent interactions overcome postcapillary shear a healthy individual, increased concentrations of micro- stress, enabling capture and rolling of leukocytes on the bial toxins and pro-inflammatory mediators induce activated endothelium. Selectin-interactions and local chemokines then activate leukocyte integrins such as * Correspondence: [email protected] lymphocyte function antigen-1 (LFA-1, CD11a/CD18) 1Department of Anesthesiology and Intensive Care Medicine, Tuebingen and macrophage antigen-1 (MAC-1, CD11b/CD18). University Hospital, Eberhard-Karls University, Hoppe-Seyler-Str. 3, Tuebingen, Local activation of integrins favours interactions with 72076, Germany Full list of author information is available at the end of the article endothelial counter-receptors, such as intercellular © 2010 Ploppa et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ploppa et al. Critical Care 2010, 14:R201 Page 2 of 13 http://ccforum.com/content/14/6/R201 adhesion molecule-1 (ICAM-1), resulting in firm Boehringer, Mannheim, Germany) and cultured in adhesion [1]. Endothelial Cell Growth Medium (EGM™,PromoCell, In contrast to local inflammation, systemic sepsis is Heidelberg, Germany) on collagen-coated rectangular characterized by profound leukocyte activation through- coverslips (Falcon Biocoat™, Becton Dickinson Labware, out the circulation [2,3]. Because organ damage is attenu- Bedford, MA, USA). Confluent HUVEC of the first pas- ated by inhibiting leukocyte-endothelial interactions, sage were used for the experiments. systemic leukocyte activation and disseminated leukocyte PMN were isolated by density gradient centrifugation adhesion are regarded essential for septic organ dysfunc- at 1,700 rpm on a discontinuous Percoll gradient with tion [4-7]. In the last few years this traditional assump- 63% and 72% Percoll in buffer (Percoll, 1.130 g/ml; tion has been challenged by the finding of an impaired Amersham Pharmacia Biotech, Uppsala, Sweden). The chemotaxis and decreased rather than increased leuko- bottom layer was collected and contaminating erythro- cyte recruitment to local sites of infection in septic indi- cytes were removed by hypotonic lysis in 10% NH4Cl on viduals despite persistent upregulation of leukocyte ice. After washing, the PMN pellet was resuspended in integrins [2,3,8-10]. Moreover, it has been recognized cold Medium 199 (Sigma, St. Louis, MO, USA) supple- that systemic hyper-inflammation often turns into hypo- mented with 50% fetal calf serum (Gibco, Mannheim, inflammation with immunosuppressive cytokine-profiles Germany) at 5 × 107/ml. To avoid assay related activa- such as increased ratios of interleukin (IL)-10 and tumor tion of PMN during rewarming, we reconstituted the necrosis factor (TNF)-a [11-13]. Similar to the phenom- PMN pellet to 106 PMN/ml just before the adhesion enon of endotoxin tolerance, endothelial sensitivity to assay in normoxic, room temperature Medium 199 only. microbial toxins becomes altered and endothelial cell Final rewarming to 37°C was achieved in the heatable adhesion molecule expression is impaired [14-17]. Para- flow chamber. doxically these changes do not seem to protect patients from the development of endothelial cell damage and Adhesion assay leukocyte-related organ dysfunction since they are most PMN adhesion to HUVEC was quantified in a parallel pronounced in those with poor prognosis [12,13]. To plate flow chamber with a laminar flow profile (Rey- provide more insight into themechanismsthatcontri- nolds number <1, Figure 1) at 37°C as previously bute to these apparently paradoxical findings, we investi- reported [18]. According to those shear forces that have gated the following questions in a flow chamber model been observed in postcapillary venules of normal and with lipopolysaccharide induced inflammation. septic individuals we varied shear stress from 3 to 0.25 First, does systemic leukocyte activation increase or dyne/cm2 [19-25]. impair leukocyte recruitment to activated endothelium PMN were perfused over HUVEC-containing cover- and what are the mechanisms during the different stages slips for 10 minutes under different conditions of LPS- of inflammation? Second, if targeted leukocyte recruit- activation. Thereafter, PMN-adhesion was determined ment to locally activated endothelium is impaired, are from 10 s video recordings of five different fields of there mechanisms that favour disseminated leukocyte view by phase contrast microscopy (20× objective; accumulation at the same time? Third, given that later DMIRB, Leica, Bensheim, Germany). PMN were defined sepsis is characterized by immunosuppression, endothe- as rolling when traveling below 50% of the velocity of lial cell damage and organ dysfunction, are there free flowing PMN in close proximity to the endothelium mechanisms, independent of the physiological immune at the given shear stress [26]. A PMN, moving less than response, that gain a leading role for the distribution of one cell diameter in 10 s was defined to be firmly adher- leukocyte accumulation? ent. To exclude sedimentation artefacts, we exposed the adherent PMN, stepwise, up to 32 dyne/cm2 after the Materials and methods end of the adhesion experiment and measured cell Endothelial cell culture and leukocyte separation detachment. Under this exposure >70% of the adherent In compliance with the Helsinki Declaration on experi- PMN remained bound. As a measure for adhesion effi- mental research on humans and after obtaining ethical ciency [27,28], the rolling fraction was calculated as: committee approval (local ethics committee, University *(No. of rolling cells) × 100)/(No. of rolling cells + No. of of Tuebingen, reference numbers 315/99 and 69/2003- firmly adherent cells). Mean rolling velocities were A) and informed consent, human umbilical venous determined from more than 25 individual velocity pro- endothelial cells (HUVEC) and polymorphonuclear neu- files for each experimental condition as derived from trophils (PMN) were derived from human umbilical customized software for image recognition (CellTracker, veins and citrated blood samples from healthy volun- C. Zanke, University of Tuebingen, Germany). teers as previously described [18]. HUVEC were har- Selectin function was determined